1. Field of the Invention
The present invention relates to a method for producing the peptide of a prenyl diphosphate synthetase, a method for producing an active type prenyl diphosphate synthetase, a DNA coding for the synthetase, a recombinant vector comprising the DNA and a transformant transformed with the vector.
2. Description of the Prior Art
An extremely wide variety of isoprenoid compounds are found in natural creatures from bacteria to higher eukaryotes. For example, steroids, carotenoids, polyprenols which are sugar carriers, quinones, tRNA modified with isopentenyladenine, prenylated proteins and the like may be enumerated. All of these isoprenoids are biosynthesized through prenyl diphosphate as an intermediate which is produced by a prenyl diphosphate synthetase (FIG. 1).
The "prenyl diphosphate synthetase" is a general term for those enzymes which catalyze a reaction that condensation-polymerizes prenyl diphosphate (an allylic primer) and 3-isopentenyl diphosphate (IPP) to produce polyprenyl diphosphate.
Prenyl diphosphate synthetases are divided into two groups. One group consists of enzymes that catalyze a condensation reaction in which the double bond formed by each condensation of IPP is of E type. The other group consists of enzymes that catalyze a condensation reaction in which the double bond formed by each condensation of IPP is of Z type. Further, the maximum length of the isoprene chain which each prenyl diphosphate synthetase can produce is fixed. Since the hydrophobic property of a product varies depending of the isoprene chain length of the product, there is great difference in the mode of requirement for the activity of enzymes. When bacterial enzymes are compared in terms of the mode of requirement, prenyl diphosphate synthetases are classified into the following four groups.
(1) Prenyl diphosphate synthetase I (E type, short chain prenyl diphosphate synthetase)
(i) Geranyl diphosphate (GPP) synthetase (Sagami, H. et al., (1978) Biochem. Biophys. Res. Commun., 85, 575) (C.sub.5.fwdarw.C.sub.10) PA1 (ii) Farnesyl diphosphate (FPP) synthetase (Takahashi, I. and Ogura, K., (1981) J. Biochem. 89, 1581; Fujisaki, S. et al., (1986) J. Biochem., 99, 1327) (C.sub.5.fwdarw.C.sub.10) PA1 (iii) Geranylgeranyl diphosphate (GGPP) synthetase (Takahashi, I. and Ogura, K., (1982) J. Biochem. 92, 1527; Sagami, H. and Ogura, K., (1981) J. Biochem., 89, 1573) (C.sub.5.fwdarw.C.sub.20) PA1 (i) Hexaprenyl diphosphate (HexPP) synthetase (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610) (C.sub.15.fwdarw.C.sub.30) PA1 (ii) Heptaprenyl diphosphate (HepPP) synthetase (Takahashi, I. et al., (1980) J. Biochem., 255, 4539) (C.sub.15.fwdarw.C.sub.35) PA1 (i) Octaprenyl diphosphate (OctPP) synthetase (Fujisaki, S. et al., (1986), J. Biochem., 99, 1327) (C..sub.15.fwdarw.C.sub.40) PA1 (ii) Nonaprenyl diphosphate (NonPP) synthetase (Sagami, H. et al., (1977) Biochemistry, 16, 4616) (C.sub.10.fwdarw.C.sub.45) Decaprenyl diphosphate (DecPP) synthetase (Ishii, K. et al., (1983) Biochem. Biophys. Res. Commun., 116, 500) (C.sub.15.fwdarw.C.sub.50) PA1 (i) Z-nonaprenyl diphosphate synthetase (Ishii, K. et al., (1986) Biochem. J., 233, 773) (C.sub.15.fwdarw.C.sub.45) PA1 (ii) Undecaprenyl diphosphate (UPP) synthetase (Takahashi, I. and Ogura, K. (1982) J. Biochem., 92, 1527; Keenman, M.V. and Allen, C.M. (1974) Arch. Biochem. Biophys., 161, 375) PA1 (iii) Dehydrodolichyl diphosphate (deDolPP) synthetase (Sagami, H. et al., (1989) Biochem. Biophys. Acta. 1002, 218) (C.sub.15.fwdarw.C.sub.85-105) PA1 a) Component A and component I are relatively high in thermostability, whereas component B and component II have thermostability as low as that of other enzymes derived from mesophiles (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610). PA1 b) There is no interchangeability between component A and component I. In other words, neither a combination of component A and component II nor a combination of component I and component B exhibits enzyme activity (Fujii, H. et al., (1983) FEBS Lett., 161, 257). PA1 c) Component B and component II are affected by SH reagent and arginine-specific reagent to lower the enzyme activity remarkably, whereas component A and component I are not affected by these reagents (Yoshida, I. et al., (1989) Biochem. Biophys. Acta, 995, 138).
The expression "C.sub.5.fwdarw.C.sub.10 " means that the subject synthetase catalyzes the synthesis from a compound with 5 carbon atoms to a compound with 10 carbon atoms (hereinafter, this indication has a similar meaning.)
(2) Prenyl diphosphate synthetase II (E type, medium chain prenyl diphosphate synthetase)
(3) Prenyl diphosphate synthetase III (E type, long chain prenyl diphosphate synthetase)
(4) Prenyl diphosphate synthetase IV (Z type, long chain prenyl diphosphate synthetase)
Prenyl diphosphate synthetase I successively condensates 3-isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) generated by isomerization of IPP as an allylic primer to thereby synthesize a short chain, totally E type prenyl diphosphate with 20 or less carbon atoms. This product serves as a precursor for steroids, carotenoids or prenylated proteins. Further, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) also serve as an allylic primer substrate for a medium- or long-chain prenyl diphosphate synthetase.
Hexaprenyl diphosphate synthetase (HexPS) and heptaprenyl diphosphate synthetase (HepPS) belong to prenyl diphosphate synthetase II. These enzymes synthesize hexaprenyl diphosphate and heptaprenyl diphosphate, respectively, without DMAPP nor GPP as a primer but using FPP as an allylic primer. The products are highly hydrophobic and serve as precursors for the side chains of menaquinones or ubiquinones in organisms having these enzymes. These prenylquinones play important roles in the respiratory chain or the electron transport system in photosynthesis.
Any member of prenyl diphosphate synthetase II is an enzyme composed of two essential proteins which do not have the catalytic activity independently. However, the enzyme has a property that in the presence of substrates for the enzyme, the two proteins associate with each other and exhibit the catalytic activity (Yoshida, I. et al., (1989) Biochem. Biophys. Res. Commun., 160, 448). In this point, the enzyme of this group is greatly different from other prenyl diphosphate synthetase.
As a microorganism producing prenyl diphosphate synthetase II, Micrococcus luteus, Bacillus subtilis and the like are known (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610; Takahashi, I. et al., (1980) J. Biol. Chem., 255, 4539). The following facts have been shown on the two components (designated "component A" and "component B") of HexPS from Micrococcus luteus B-P 26 and on the two components (designated "component I" and "component II") of HepPS of Bacillus subtilis.
Octaprenyl diphosphate synthetase (OctPS), nonaprenyl diphosphate synthetase (NonPS) and the like belong to prenyl diphosphate synthetase III. Like prenyl diphosphate synthetase I, these enzymes are a homodimeric protein composed of identical subunits. They exhibit the catalytic activity by themselves. However, in order to maintain the turnover as a catalyst, they require a proteinaceous factor which removes hydrophobic products from their active site (Ohnuma, S. et al., (1991) J. Biol. Chem., 266, 23706). This activator is interchangeable and exhibits activating action against any enzyme belonging to prenyl diphosphate synthetase III (Ohnuma, S. et al., (1991) J. Biol. Chem., 266, 23706).
Enzymes belonging to prenyl diphosphate synthetase IV condensate IPP in the Z-structual form to synthesize polyprenyl diphosphate of E-and-Z mixed type using a short-chain prenyl diphosphate (GPP, FPP) as a primer substrate. Bacterial undecaprenyl diphosphate synthetase (UPS) and eukaryotic dehydrodolichyl diphosphate synthetase (deDolPS) are included in this group. A large number of these enzymes are a membrane-bound protein and when solubilized with a surfactant or the like, they require the addition of a surfactant such as Triton X-100 for the manifestation of their activity in almost all cases (Takahashi, I and Ogura, K. (1982) J. Biochem., 92, 1527; Allen, C. M. and Muth, J. D. (1977) Biochemistry, 16, 2908). Additionally, the activator common in prenyl diphosphate synthetase III is ineffective against UPS. It is considered that this fact is because hydrophobic environment of a membrane is essential for the manifestation of the enzyme activity.
The most part of the above-described information has been obtained from experiments using those enzymes extracted and purified from a solution of disrupted cells. In order to clarify a more detailed enzyme reaction mechanism, not only the primary structure but also the crystal structure of enzyme proteins should be analyzed. For this purpose, the cloning of genes coding for these enzyme proteins is indispensable.
Actually, prenyl diphosphate synthetase genes such as FPS and GGPS have been cloned recently one by one (FPP synthetases: Koyama, T. et al., (1993) J. Biochem., 113, 355; Fujisaki, S. et al., (1990) J. Biochem., 108, 995; Anderson, M. A. et al., (1989) J. Biol. Chem., 264, 19176; Clarke, C.F. et al., (1987) Mol. Cell. Bio., 7, 3138; Wilkin, D. J. et al., (1990) J. Biol. Chem., 265, 4607; GGPP synthetases: Carattoli, A. et al., (1991) J. Biol. Chem., 266, 5854; Armstrong, G.A. et al., (1990) Proc. Natl. Acad. Sci. USA, 87, 9975; Math, S. K. et al., (1992) Proc. Natl. Acad. Sci. USA, 89, 6761; Misawa, N. et al., (1990) J. Bacteriol., 172, 6704). With respect to HexPP synthetase, a gene coding for one of the two components (corresponding to "component B" described previously) has been cloned by an experiment on complementarity in yeast. However, the two components of this synthetase are necessary for the manifestation of the activity, as described previously. Therefore, it cannot be said that a perfect cloning of the gene coding for the enzyme of active type has been performed (HexPP synthetase: Ashby, M. M. and Edwards, P. A. (1990) J. Biol. Chem., 265, 13157).
The present inventor has compared the primary structures of the above-mentioned enzymes based on the base sequences for their genes. As a result, it has become clear that prenyl diphosphate synthetases have 7 regions in which the amino acid sequence has been relatively preserved beyond the difference in chain length or organism species (Koyama, T. et al., (1993) J. Biochem. 113, 355-363). Since these regions are preserved in a group of enzymes which catalyze substantially the same reaction, they are believed to have an important role in the catalytic function. On the other hands, it is predicted that non-preserved regions have a portion defining the chain length, a portion involved in the difference in the mode of manifestation of the enzymatic function, and the like. However, at present, the number of cloned genes of prenyl diphosphate synthetases having different chain length is too small to find out the existence of such portions from comparison of primary structures.
From the viewpoint of the manifestation of enzymatic function, enzymes belonging to prenyl diphosphate synthetase II are greatly different from other prenyl diphosphate synthetases, as described previously. They are characterized by being composed of two proteins (heterodimeric type), each of which does not have a catalytic function alone but which associate with each other in the presence of a substrate to exhibit a catalytic function.
Substances synthesized by these heterodimeric prenyl diphosphate synthetases are precursors of those substances such as vitamin K and ubiquinones which exist universally in organisms and, thus, they are important physiologically active substances. Therefore, they are of high utility value. Furthermore, the prenyl diphosphate produced by a heterodimeric prenyl diphosphate synthetase is industrially extremely useful since the chain length and structural isomers thereof can be strictly controlled. Thus, the expression of such a synthetase in large quantity is needed.
Accordingly, it is desired to isolate genes coding for the two proteins of an enzyme belonging to prenyl diphosphate synthetase II, to express the genes separately and thereby to produce the proteins in large quantity.